This invention relates to specific binding members for a fetal isoform of fibronectin, ED—B, which is also expressed in the developing neovasculature of tumors, as demonstrated both by immunocytochemistry and by targeting of tumors in vivo. It also relates to materials and methods relating to such specific binding members.
The primary aim of most existing forms of tumor therapy is to kill as many constituent cells of the tumor as possible. The limited success that has been experienced with chemotherapy and radiotherapy relates to the relative lack of specificity of the treatment and the tendency to toxic side-effects on normal tissues. One way that the tumor selectivity of therapy may be improved is to deliver the agent to the tumor through a binding protein, usually comprising a binding domain of an antibody, with specificity for a marker antigen expressed on the surface of the tumor but absent from normal cells. This form of targeted therapy, loosely termed ‘magic bullets’, has been mainly exemplified by monoclonal antibodies (mAbs) from rodents which are specific for so-called tumor-associated antigen expressed on the cell surface. Such mAbs may be either chemically conjugated to the cytotoxic moiety (for example, a toxin or a drug) or may be produced as a recombinant fusion protein, where the genes encoding the mAb and the toxin are linked together and expressed in tandem.
The ‘magic bullet’ approach has had limited, although significant, effect in the treatment of human cancer, most markedly in targeting tumors of lymphoid origin, where the malignant cells are most freely accessible to the therapeutic dose in the circulation. However, the treatment of solid tumors remains a serious clinical problem, in that only a minute proportion of the total cell mass, predominantly the cells at the outermost periphery of the tumor, is exposed to therapeutic immunoconjugates in the circulation; these peripheral targets form a so-called ‘binding site barrier’ to the tumor interior (Juweid et al, 1992, Cancer Res. 52 5144-5153). Within the tumor, the tissue architecture is generally too dense with fibrous stroma and closely packed tumor cells to allow the penetration of molecules in the size range of antibodies. Moreover, tumors are known to have an elevated interstitial pressure owing to the lack of lymphatic drainage, which also impedes the influx of exogenous molecules. For a recent review of the factors affecting the uptake of therapeutic agents into tumors, see Jain, R (1994), Sci. Am. 271 58-65.
Although there are obvious limitations to treating solid tumors through the targeting of tumor-associated antigens, these tumors do have a feature in common which provides an alternative antigenic target for antibody therapy. Once they have grown beyond a certain size, tumors are universally dependent upon an independent blood supply for adequate oxygen and nutrients to sustain growth. If this blood supply can be interfered with or occluded, there is realistic potential to starve thousands of tumor cells in the process. As a tumor develops, it undergoes a switch to an angiogenic phenotype, producing a diverse array of angiogenic factors which act upon neighboring capillary endothelial cells, inducing them to proliferate and migrate. The structure of these newly-formed blood vessels is highly disorganized, with blind endings and fenestrations leading to increased leakiness, in marked contrast to the ordered structure of capillaries in normal tissue. Induction of angiogenesis is accompanied by the upregulation of expression of certain cell surface antigens, many of which are common to the vasculature of normal tissues. Identifying antigens which are unique to neovasculature of tumors has been the main limiting factor to developing a generic treatment of solid tumors through vascular targeting. The antigen which is the subject of the present invention addresses this problem directly.
During tumor progression, the extracellular matrix of the surrounding tissue is remodelled through two main processes: (1) the proteolytic degradation of extracellular matrix components of normal tissue and (2) the de novo synthesis of extracellular matrix components by both tumor cells and by stromal cells activated by tumor-induced cytokines. These two processes, at steady state, generate a “tumoral extracellular matrix”, which provides a more suitable environment for tumor progression and is qualitatively and quantitatively distinct from that of normal tissues. Among the components of this matrix are the large isoforms of tenascin and fibronectin (FN); the description of these proteins as isoforms recognizes their extensive structural heterogeneity which is brought about at the transcriptional, post-transcriptional and post-translational level (see below). It is one of the isoforms of fibronectin, the so-called B+ isoform (B—FN), that is the subject of the present invention.
Fibronectins (FN) are multifunctional, high molecular weight glycoprotein constituents of both extracellular matrix and body fluids. They are involved in many different biological processes such as the establishment and maintenance of normal cell morphology, cell migration, homeostasis and thrombosis, wound healing and oncogenic transformation (for reviews see Alitalo et al., 1982; Yamada, 1983; Hynes, 1985; Ruoslahti et al., 1998; Mynes, 1990; Owens et al., 1986). Structural diversity of FnS is brought about by alternative splicing of three regions (ED—A, ED—B and IIICS) of the primary FN transcript (Hynes, 1985; Zardi et al., 1987) to generate at least 20 different isoforms, some of which are differentially expressed in tumor and normal tissue. As well as being regulated in a tissue- and developmentally-specific manner, it is known that the splicing pattern of FN-pre-mRNA is deregulated in transformed cells and in malignancies (Castellani et al., 1986; Borsi et al, 1987; Vartio et al., 1987, Zardi et al, 1987; Barone et al, 1989; Carnemolla et al, 1989; Oyama et al, 1989, 1990; Borsi et al, 1992b). In fact, the FN isoforms containing the ED—A, ED—B and IIICS sequences are expressed to a greater extent in transformed and malignant tumor cells than in normal cells. In particular, the FN isoform containing the ED—B sequence (B+ isoform), is highly expressed in fetal and tumor tissues as well as during wound healing, but restricted in expression in normal adult tissues (Norton et al, 1987; Schwarzbauer et al, 1987; Gutman and Kornblihtt, 1987; Carnemolla et al, 1989; ffrench-Constant et al, 1989; ffrench-Constant and Hynes, 1989; Laitinen et al, 1991. ) B+ FN molecules are undetectable in mature vessels, but unregulated in angiogenic blood vessels in normal (e.g. development of the endometrium), pathologic (e.g. in diabetic retinopathy) and tumor development (Castelliani et al, 1994).
The ED—B sequence is a complete type III-homology repeat encoded by a single exon and comprising 91 amino acids. In contrast to the alternatively spliced IIICS isoform, which contains a cell type-specific binding site, the biological function of the A+ and B+ isoforms is still a matter of speculation (Humphries et al., 1986).
The presence of B+ isoform itself constitutes a tumor-induced neoantigen, but in addition, ED—B expression exposes a normally cryptic antigen within the type III repeat 7 (preceding ED—B); since this epitope is not exposed in FN molecules lacking ED—B, it follows that ED—B expression includes the expression of neoantigens both directly and indirectly. This cryptic antigenic site forms the target of a monoclonal antibody (mAb) named BC-1 (Carnemolla et al, 1992). The specificity and biological properties of this mAb have been described in EP 0 344 134 B1 and it is obtainable from the hybridoma deposited at the European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK under the number 88042101. The mAb has been successfully used to localize the angiogenic blood vessels of tumors without crossreactivity to mature vascular endothelium, illustrating the potential of FN isoforms for vascular targeting using antibodies.
However, there remain certain caveats to the specificity of the BC-1 mAb. The fact that BC-1 does not directly recognize the B+ isoform has raised the question of whether in some tissues, the epitope recognized by BC-1 could be unmasked without the presence of ED—B and therefore lead indirectly to unwanted crossreactivity of BC-1. Furthermore, BC-1 is strictly specific for the human B+ isoform, meaning that studies in animals on the biodistribution and tumor localization of BC-1 are not possible. Although polyclonal antibodies to recombinant fusion proteins containing the B+ isoform have been produced (Peters et al, 1995), they are only reactive with FN which has been treated with N-glycanase to unmask the epitope.
A further general problem with the use of mouse monoclonal antibodies is the human anti-mouse antibody (HAMA) response (Schroff et al, 1985; Dejager et al, 1988). HAMA responses have a range of effects, from neutralization of the administered antibody leading to a reduced therapeutic dose, through to allergic responses, serum sickness and renal impairment.
Although polyclonal antisera reactive with recombinant ED—B have been identified (see above), the isolation of mAbs with the same specificity as BC-1 following immunization of mice has generally proved difficult because human and mouse ED—B proteins show virtually 100% sequence homology. The human protein may therefore look like a self-antigen to the mouse which then does not mount an immune response to it. In fact, in over ten years of intensive research in this field, only a single mAb has been identified with indirect reactivity to the B+ FN isoform (BC-1), with none recognizing ED—B directly. It is almost certainly significant that the specificity of BC-1 is for a cryptic epitope exposed as a consequence of ED—B, rather than for part of ED—B itself, which is likely to be absent from mouse FN and therefore not seen as “self” by the immune system of the mouse.
Realization of the present invention has been achieved using an alternative strategy to those previously used and where prior immunization with fibronectin or ED—B is not required: antibodies with specificity for the ED—B isoform have been obtained as single chain Fvs (scFvs) from libraries of human antibody variable regions displayed on the surface of filamentous bacteriophage (Nissin et al., 1994; see also WO92/01047, WO92/20791, WO93/06213, WO93/11236, WO93/19172).
We have found using an antibody phage library that specific scFvs can be isolated both by direct selection on recombinant FN-fragments containing the ED—B domain and on recombinant ED—B itself when these antigens are coated onto a solid surface (“panning”). These same sources of antigen have also been successfully used to produce “second generation” scFvs with improved properties relative to the parent clones in a process of “affinity maturation”. We have found that the isolated scFvs react strongly and specifically with the B+ isoform of human FN without prior treatment with N-glycanase.
In anti-tumor applications the human antibody antigen binding domains provided by the present invention have the advantage of not being subject to the HAMA response. Furthermore, as exemplified herein, they are useful in immunohistochemical analysis of tumor tissue, both in vitro and in vivo. These and other uses are discussed further herein and are apparent to the person of ordinary skill in the art.